1. Field of Invention
The present invention relates to telomerase, a ribonucleoprotein enzyme involved in telomere DNA synthesis, and provides assays and protocols for identifying and measuring telomerase activity and correlating that activity with disease conditions. The invention provides methods and compositions relating to the fields of molecular biology, chemistry, pharmacology, and medical diagnostic and prognostic technology.
2. Description of Related Disclosures
Telomeres are specialized structures at the ends of eukaryotic chromosomes and appear to function in chromosome stabilization, positioning, and replication (Blackburn and Szostak, 1984, Ann. Rev. Biochem. 53:163-194; Zakian, 1989, Ann. Rev. Genetics 23:579-604; Blackburn, 1991 Nature 350:569-573). In all vertebrates, telomeres consist of hundreds to thousands of tandem repeats of 5'-TTAGGG-3' sequence and associated proteins (Blackburn, 1991; Moyzis et al., 1988, Proc. Natl. Acad. Sci. 85:6622-6626). Southern blot analysis of chromosome terminal restriction fragments (TRF) provides the composite lengths of all telomeres in a cell population (Harley et al., 1990, Nature 345:458-460; Allsopp et al., 1992, Proc. Natl. Acad. Sci. USA 89:10114-10118; Vaziri et al., 1993, Am. J. Human Genetics 52:661-667). In all normal somatic cells examined to date, TRF analysis has shown that the chromosomes lose about 50-200 nucleotides of telomeric sequence per cell division, consistent with the inability of DNA polymerase to replicate linear DNA to the ends (Harley et al., 1990; Allsopp et al., 1992; Vaziri et al., 1993; Watson, 1972, Nature New Biology 239:197-201).
This shortening of telomeres has been proposed to be the mitotic clock by which cells count their divisions (Harley, 1991, Mut. Res. 256:271-282), and a sufficiently short telomere(s) may be the signal for replicative senescence in normal cells (Allsopp et al., 1992; Vaziri et al., 1993; Hastie et al., 1990, Nature 346:866-868; Lindsey et al., 1991, Mut. Res. 256:45-8; Wright and Shay, 1992, Trends Genetics 8:193-197). In contrast, the vast majority of immortal cells examined to date show no net loss of telomere length or sequence with cell divisions, suggesting that maintenance of telomeres is required for cells to escape from replicative senescence and proliferate indefinitely (Counter et al., 1992, EMBO 11:1921-1929; Counter et al., 1994, Proc. Natl. Acad. Sci. USA 91:2900-2940).
Telomerase, a unique ribonucleoprotein DNA polymerase, is the only enzyme known to synthesize telomeric DNA at chromosomal ends using as a template a sequence contained within the RNA component of the enzyme (Greider and Blackburn, 1985, Cell 43:405-413; Greider and Blackburn, 1989, Nature 337:331-337; Yu et al., 1990, Nature 344:126-132; Blackburn, 1992, Ann. Rev. Biochem. 61:113-129). With regard to human cells and tissues, telomerase activity has been identified in immortal cell lines and in ovarian carcinoma but has not been detected in mortal cell strains or in normal non-germline tissues (Counter et al., 1992; Counter et al., 1994; Morin, 1989, Cell 59:521-529). Together with TRF analysis, these results suggest telomerase activity is directly involved in telomere maintenance, linking this enzyme to cell immortality.
Scientists have therefore proposed that senescence, or mortality stage 1 (M1), occurs when there are on average several kilobases of telomeric repeats remaining and involves the anti-proliferative actions of tumor suppressor gene products such as pRb and p53 (Shay et al., 1993, Oncogene 8:1407). Mutations in these genes, or expression of viral transforming genes that block the action of these genes, permit cells to undergo additional divisions in the absence of telomerase until the telomeres reach a critically short length at crisis, or mortality stage 2 (M2). See Wright et al., 1989, Mol. Cell. Biol. 9:3088. At crisis, there is destabilization of chromosomes resulting in an increase in the frequency of dicentric chromosomes and cessation of cell proliferation. Development of an immortalized cell line after crisis is dependent on expression of telomerase activity. After crisis, telomerase can stabilize telomere length and permit indefinite cell division (Blackburn, 1994, Cell 77:621).
Methods for detecting telomerase activity, as well as for identifying compounds that regulate or affect telomerase activity, together with methods for therapy or diagnosis of cellular senescence and immortalization by controlling or measuring telomere length and telomerase activity, have also been described. See PCT patent publication No. 93/23572, published Nov. 25, 1993, incorporated herein by reference. The identification of compounds affecting telomerase activity provides important benefits to efforts at treating human disease. Compounds that inhibit telomerase activity can be used to treat cancer, as cancer cells express and require telomerase activity for immortality, and normal human somatic cells do not express telomerase activity at detectable levels. Compounds that stimulate or activate telomerase activity can be used to treat age-related diseases and other conditions relating to cell senescence.
New and improved methods for screening to identify compounds that modulate telomerase activity as well as for measuring telomerase activity in a sample have been developed; see, e.g., U.S. patent application Ser. No. 08/315,214, filed Sep. 28, 1994 inventors Calvin B. Harley, Nam Woo Kim, and Scott Weinrich, incorporated herein by reference. Other methods for assaying telomerase activity in cell samples rely on the incorporation of radioactively labeled nucleotides into a telomerase substrate (Morin, 1989). The conventional assay uses an oligonucleotide substrate, a radioactive deoxyribonucleoside triphosphate (dNTP) for labeling, and gel electrophoresis for resolution and display of products. Because telomerase stalls and can release the DNA after adding the first G in the 5'-TTAGGG-3' telomeric repeat, the characteristic pattern of products on the gel is a six nucleotide ladder of extended oligonucleotide substrates. The phase of the repeats depends on the 3'-end sequence of the substrate; telomerase recognizes where the end is in the repeat and synthesizes accordingly to yield contiguous repeat sequences. Although telomeric sequence oligonucleotides are efficient in vitro substrates, telomerase will also synthesize repeats using substrates comprising non-telomeric DNA sequences.
Using such methods, scientists have found that the presence of telomerase activity in somatic tissues is positively correlated with cancer. There remains a need, however, for diagnostic methods that enable the physician to correlate the presence of telomerase activity in a sample with the likelihood that a particular type of cancer is likely to be invasive or metastasize or recur, to assess whether cancer cells remain in a patient after surgery, chemotherapy, or other treatments, and to diagnose a patient's predisposition to cancer, and this invention meets that need.